The Neurotrophin Family
Neurotrophins are dimeric peptide hormones. The first member of the neurotrophin family to be discovered was nerve growth factor (NGF), which plays an important role in processes such as the development of sensory and sympathetic neurons of the peripheral nervous system (Levi-Montalcini, R. and Angeleeti, P. U, Physiol. Rev. 48, 534-569 (1968)). The next member of the neurotrophin family to be isolated was brain-derived neurotrophic factor (BDNF), also referred to as neurotrophin-2 (NT-2), the sequence of which was published by Leibrock, J. et al. in 1989 (Nature 341, 149-152). In 1990 several groups identified a neurotrophic factor originally called neuronal factor (NF), now referred to as neurotrophin-3 (NT-3) (Ernfors et al., Proc. Natl: Acad. Sci. USA 87, 5454-5458 (1990); Hohn et al., Nature 344, 339; Maisonpierre et al., Science 247, 1446; Rosenthal et al., Neuron 4, 767; Jones and Reichardt, Proc. Natl. Acad. Sci. USA 87, 8060-8064; Kaisho et al., FEBS Lett. 266, 187). Neurotrophins-4 and -5 were then added to the family (Neuron 6, 845-858 (1991); Berkmeier, L. R. et al., Neuron 7, 857-866 (1991); Ip et al., Proc. Natl. Acad. Sci. USA 89, 3060-3064 (1992)). The effects of neurotrophins depend upon their levels of availability, their affinity of binding to transmembrane receptors and the downstream signalling cascades that are stimulated after receptor activation.
Receptors for the Neurotrophin Family
In a similar way to other polypeptide growth factors, neurotrophins affect their target cells through interactions with cell surface receptors. According to current knowledge, neurotrophins bind to two discrete receptor types which can be distinguished pharmacologically: the Trk and p75NTR neurotrophin receptors. p75NTR is a member of the Fas/tumour necrosis factor (TNF) receptor family, and can interact with all the mammalian members of the neurotrophin family with equal affinities (Rodriguez-Tebar et al. 1990, Neuron 4:487-492; Barker and Murphy, 1992, Mol. Cell. Biochem. 100:1-15). Cells expressing TrkA, a tyrosine kinase receptor originally identified as a human oncogene (Mltin-Zanca et al, Nature 319:743-748) bind solely to NGF and exhibit significantly slower dissociation kinetics (Jing et al. 1992, Neurol. 9:1067-1079; Loeb and Greene, 1993, Neuroscience 13:2919-2929). BDNF binds the TrkB receptor only, but NT-3 can bind all three Trk (A, B and C) receptors, with a preference for TrkC. NT-4/5 can bind both TrkA and TrkB (Ip et al. PNAS 89:3060-3064; Klein et al. Neuron 9:947-956). NT-7 does not interact with TrkB or TrkC but can however induce tyrosine phosphorylation of TrkA, indicating a similar receptor specificity as NGF (Nilsson et al., FEBS Lett (1998) March 13; 424(3):285-90). Recombinant purified NT-6 also has a spectrum of actions similar to NGF but with a lower potency (Gotz et al., Nature (1994) November 17; 372(6503):266-9).
The Neurotrophin Family: Precursor Proteins
The biology of the neurotrophin family is complex: the neurotrophins are synthesised intracellularly as 30-35 kDa precursor proteins, containing a signal peptide a pro-domain and glycosylation sites. During processing precursor proteins are also cleaved at a dibasic cleavage site by the calcium-dependent serine protease furin and other members of the prohormone convertase family, within the Golgi apparatus. The 12-14 kDa C-terminal product of this cleavage is the mature, biologically active neurotrophin (Seidah et al, Biochem. J. (1996) 314:951-960).
Clinically Relevant Roles of the Neurotrophin Family
Neurotrophins are of clinical interest as they play an important role in neuronal cell survival and differentiation (Thoenen 1991, Trends Neurosci. 14: 165-170; Raffioni et al. 1991, Ann. Rev. Biochem. 62:823-850; Chao, 1992, Neuron 9:583-593; Barbacid 1993, Oncogene 8:2033-2042). Trk receptors transmit signals promoting neuronal survival, whereas p75NTR can induce neuronal apoptosis as well as neuronal survival depending on co-expression of Trk (Miller et al., Cell. Mol. Life. Sci. 58:1045-1053 (2001)). Certainly, it has been demonstrated that activation of TrkA receptors can negate the proapoptotic effect of p75NTR in some but not all tissues (Yoon et al., J. Neurosci. (1998) 18:3273-3281; Volosin et al., J. Neurosci. (2006) 26:7756-7766).
It has been demonstrated that the propeptides of neurotrophins play important biological roles: at least three neurotrophin precursor proteins, proNGF, pro-BDNF and proNT-3 and their proteolytically processed and mature counterpart, NGF, BDNF, NT-3 products differentially activate pro- and anti-apoptotic cellular responses through preferential activation of p75NTR and Trk receptors, respectively—pro-NGF having enhanced affinity for p75NTR receptors and a reduced affinity for Trk receptors relative to the mature forms of NGF. Indeed, it has been demonstrated that pro-NGF induces p75NTR-dependent apoptosis in cultured neurons with minimal activation of TrkA-mediated differentiation or survival (Lee et al., Science (2001), 294:1945-1948).
Furthermore, neurotrophins are of clinical interest as it is known that both up-regulation of neurotrophins and increased p75NTR expression occur under pathological and inflammatory conditions, especially after nerve injury and damage to the vascular system. Indeed, Soilu-Hanninen et al. have demonstrated that the proapoptotic functions of p75NTR are directly implicated in injury-induced apoptosis (Soilu-Hanninen et al., J. Neurosci. 19:4824-4838 (1999)). Recently, it was also demonstrated that proNGF induces p75 mediated death of oligodendrocytes and corticospinal neurons following spinal cord injury (Beatty et al., Neuron (2002), vol. 36, pp. 375-386; Giehl et al, Proc. Natl. Acad. Sci USA (2004), vol. 101, pp 6226-30) and death of basal forebrain neurons in response to kainic acid-induced seizures (Volosin et al, J. Neuroscience (2006), vol. 26, pp 7756-7766).
It has been hypothesized that an imbalance between the precursor and mature form of neurotrophic factors is responsible for the degeneration of selective neuronal populations as it occurs in Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis, and that application of corresponding neurotrophic factor might prevent neuronal degeneration [Appel, S. H., “A unifying hypothesis for the cause of amyotrophic lateral sclerosis, parkinsonism, and Alzheimer's disease,” Ann. Neurol. 10:499-505 (1981), Cunello C and Bruno M. A., Neurochem. Res. (2007) 32:1041-45].
Another reason for interest in targeting neurotrophin pathways for therapy is that studies have provided supporting evidence for the involvement of neurotrophins in depression and antidepressant action (Duman et al. Arch Gen Psychiatry (1997) 54:597-606); for instance infusion of BDNF into the hippocampus has produced an antidepressant effect in two behavioural models of depression (Shirayama et al. (2002), J Neurosci 22(8): 3251-3261). Moreover, a single nucleotide polymorphism in the bdnf gene leading to a valine (Val) to methionine (Met) substitution at codon 66 in the pro-domain (BDNFMet) was found to be associated with increased susceptibility in humans heterozygous for the polymorphism to neuropsychiatric disorders including Alzheimer's disease, Parkinson's disease, depression, and bipolar disorder (Chen et al, J. Neuroscience (2005), vol. 25:6156-6166; Kuipers and Bramham Curr. Opin. Drug Discov. Devel. (2006) 9(5):580-6; Bath and Lee, Cogn. Affect. Behav. Neurosci (2006) 1:79-85). In addition, humans heterozygous for BDNFMet were shown to have memory impairments (Egan et al, Cell (2003)1 vol. 112, pp 257-269).
The Vps10p-Domain Receptor Family
Sortilin (or NTR-3 or GP95), (SEQ ID NO. 1) is a type I membrane receptor expressed in a number of tissues, including the brain, spinal cord, testis and skeletal muscle (Petersen et al., J. Biol. Chem., 272:3599-3605 (1997); Herman-Borgmeyer et al., Mol. Brain. Res., 65:216-219 (1999)). Sortilin belongs to a family of receptors comprising Sortilin, SorLA (Jacobsen et al., J. Biol. Chem., 271:31379-31383 (1996)), SorCS1, SorCS2 and SorCS3. All the receptors in this family share the structural feature of an approximately 600-amino acid N-terminal domain with a strong resemblance to each of the two domains which constitute the luminal portion of the yeast sorting receptor Vps10p (Marcusson, E. G., et al., Cell, 77:579-586 (1994)). The Vps10p-domain includes a C-terminal segment containing 10 conserved cysteines and an N-terminal propeptide of 40-80 amino acids.
In Sortilin, the propeptide exhibits high affinity binding to the fully processed receptor. Prevention of propeptide cleavage essentially inhibits ligand binding to Sortilin, indicating that the propeptide sterically hinders ligands from gaining access to their binding sites on the receptor (Petersen et al., EMBO J., 18:595-604, 1999).
Some progress has been made as to an understanding of the role of this family: there is evidence suggesting that Sortilin at least contains YXXφ and dileucine motifs, conforming to potent signals for Golgi-endosome sorting (Nielsen et al., EMBO 20(9):2180-2190). It is probable that the other members of the family may also fulfil a similar “sorting” function, not least because they all exhibit homology to Vps10p, the sorting receptor for carboxypeptidase Y (CPY) in yeast. Only a small proportion of Sortilin receptors are present on the cell surface (Mazella et al. J. Biol. Chem. (1998) 273, 26273-26276; Morris et al. J. Biol. Chem. (1998) 273:3582-3587), although expression on the surface membrane can be upregulated by stimuli including insulin in 3T3-L1 adipocytes (Morris et al. J. Biol. Chem. (1998) 273:3582-3587) and neurotensin in embryonic neurons (Chabry et al., J. Biol. Chem. (1993), 286:17138-17144).
Inhibiting Proneurotrophin Activity: the Current State of the Art
Certainly, current understanding of the biological roles of neurotrophins makes the neurotrophin family an attractive target for therapeutic intervention, and some methods for modulation of neurotrophin activity are known:
Mehar M et al., Eur. J. Neruosci. (2006) 24:1575-1580 and Massa S. M. et al, J. Neurosci. (2006), 26:5288-5300 describe how p75 can be used as a drug target to interfere with death-induction following ligand (e.g. proNGF) binding to p75.
WO 2004/056385 discloses general methods for inhibiting binding between Vps10-p domain receptors and neurotrophins/pro-neurotrophins but fails to teach the specific binding site.